This invention relates to plant molecular biology in general, and in particular, to nucleic acid sequences which regulate the internal and 3xe2x80x2-proximal gene expression in polycistronic mRNA transcripts. This invention will enable the control of transgene expression through the generation of polycistronic fusion mRNAs in which all the genes are translationally active due to the presence of the IRESmp element(s).
According to the ribosome scanning model, traditional for most eukaryotic mRNA, the 40S ribosomal subunit binds to the 5xe2x80x2-cap and moves along the nontranslated 5xe2x80x2-sequence until it reaches an AUG codon (Kozak (1986) Adv.Virus Res. 31:229-292; Kozak (1989) J.Mol.Biol. 108: 229-241). Although for the majority of eukaryotic mRNAs only the first open reading frame (ORF) is translationally active, there are different mechanisms by which mRNA may function polycistronically (Kozak (1986) Adv.Virus Res. 31:229-292). If the the first AUG has unfavorable sequence context, 40S subunits may bypass it and initiate at downstream AUG codon (leaky scanning mechanism). Termination-reinitiation has also been suggested to explain the initiation of translation of functionally dicistronic eukaryotic mRNAs (Kozak (1989) J.Mol.Biol. 108: 229-241). Another mechanism for discontinuous ribosome migration (xe2x80x9cshuntingxe2x80x9d) on mRNA has been recently proposed for cauliflower mosaic virus (CaMV) 35S RNA (Futerrer et al.(1993) Cell 73: 789-802).
In contrast to the majority of eukaryotic mRNAs, the initiation of translation of picornavirus RNAs takes place by an alternative mechanism of internal ribosome entry. A picornaviral 5xe2x80x2-nontranslated region (5xe2x80x2NTR) contains a so-called internal ribosome entry site (IRES) or ribosome landing pad (Pelletier and Sonennberg (1988) Nature 334: 320-325; Molla. et al. (1992) Nature 356; 255-257) which is folded into a complex secondary structure and contains a pyrimidine-rich tract followed by an AUG codon (Agol (1991) Adv.Virus Res. 40: 103-180; Wimmer et al. (1993) Annu.Rev.Genet.27: 353-436; Sonennberg and Pelletier (1989) BioEssays 11: 128-132). Internal ribosome entry has also been reported for other viral (Le et al. (1994) Virology 198: 405-411; Gramstat et al. (1994) Nucleic Acid Res. 22: 3911-3917) and cellular (Oh et al. (1992) Gen Dev. 6: 1643-1653) RNAs.
It is important to emphasize that the picornavirus and other known IRESes are not active in the plant cell systems.
The genome of tobamoviruses (TMV UI is the type member) contains four large ORFs. In vitro translational experiments have shown that the two components of the replicase (the 130K and its read-through 183K proteins) are translated directly from the genomic RNA (Pelham and Jackson (1976) Eur.J.Biochem 67: 247-256). The other two proteins (30K movement protein, MP, and coat protein, CP) are translated from two individual subgenomic RNAs (sgRNAs). Two structurally dicistronic I2 sgRNA is translated to give the 30K MP, while its 3xe2x80x2-terminal CP gene is silent and a monocistronic sgRNA codes the CP (Palukaitis and Zaitlin (1986) in The Plant Viruses, eds. Van Regenmortel and M.Fraenkel-Conrat, 2: 105-131, Plenum Press).
Recently a new tobamovirus, crTMV, has been isolated from Oleracia officinalis L. plants and the genome has been sequenced (6312 nucleotides) (Dorokhov et al. (1993) Doklady of Russian Academy of Sciences 332: 518-522; Dorokhov et al. (1994) FEBS Lett. 350: 5-8). A peculiar feature of crTMV is its ability to infect systemically the members of Cruciferae family. The crTMV RNA contains four ORFs encoding the proteins of 122K (ORF1), 178K (ORF2), the read through product of 122K, 30K MP (ORF3) and 17K CP (ORF4). Unlike other tobamoviruses, the coding regions of the MP and CP genes of crTMV overlap for 25 codons, i.e. 5xe2x80x2 of the CP coding region are sequences encoding MP.
It has been shown that unlike the RNA of typical tobamoviruses, translation of the 3xe2x80x2-proximal CP gene of crTMV RNA occurs in vitro and in planta by mechanism of internal ribosome entry which is mediated by a specific sequence element, IREScp (Ivanov et al. (1997) Virology 231, in press). In that work three types of synthetic dicistronic RNA transcripts were constructed and translated in vitro: (i) xe2x80x9cMP-CP-3xe2x80x2NTRxe2x80x9d transcripts contained MP gene, CP gene and the 3xe2x80x2-nontranslated region (NTR) of crTMV. These constructs were structurally equivalent to dicistronic subgenomic RNAs produced by tobamoviruses in vivo. (ii) xe2x80x9cxcex94NPT-CPxe2x80x9d transcripts contained partially truncated neomycine phosphotransferase I gene and CP gene. (iii) xe2x80x9cCP-GUSxe2x80x9d transcripts contained the first CP gene and the gene of E.coli (xcex2-glucuronidase (GUS) at the 3xe2x80x2-proximal position.
The results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained IREScp promoting internal initiation of translation in vitro. Dicistronic IREScp containing chimeric mRNAs with the 5xe2x80x2-terminal stem-loop structure preventing translation of the first gene (MP, xcex94NPT or CP), expressed the CP or GUS genes despite their 3xe2x80x2-proximal localization. The capacity of crTMV IREScp for mediating internal translation distinguishes this tobamovirus from the well known type member of the genus, TMV UI. The equivalent 148-nt sequence from TMV RNA was uncapable of mediating internal translation. Two mutants were used to study structural elements of IREScp. It was concluded that integrity of IREScp was essential for internal initiation. The RNA analysis of IREScp revealed the polypurine-rich stretch and stem-loop structure.
The crTMV provides a new example of internal initiation of translation, which is markedly distinct from IRESes shown for picornaviruses and other viral and eukaryotic mRNAs.
In order to show that the IREScp is active not only in vitro, but also in vivo two approaches were used: i) constructing of the transgenic rapeseed plants (Brassica napus L.) containing in its genome the crTMV cDNA including MP, CP genes and 3xe2x80x2NTR; ii) the particle gun bombardment of tobacco plant leaves with the cDNA construct xe2x80x9cCP-IREScp-GUSxe2x80x9d under the control of CaMV 35S promoter and terminator. Both approaches show that IREScp is active in plants (Ivanov et al., results not published).
A primary object of this invention is to provide a method which will enable to express simultaneously desired genes in vitro and in planta. This object is to be accomplished by utilising crTMV RNA sequences upstream of MP gene is termed here as IRESmp. The method of this invention involves the construction of recombinant DNA molecule which comprises of a transcriptional promoter, the first plant-exspressible gene linked to the said transcriptional promoter, IRESmp located 3xe2x80x2 to the first gene and the second plant-expressible gene located 3xe2x80x2 to IRESmp such that the second gene is placed under the translational control of IRESmp. The primary chimeric continuous RNA transcript in positive sense polarity is produced by the transformed cells from plant-expressible promoter. The expression of the first gene takes place by direct translation of the 5xe2x80x2-proximal gene of this mRNA but the translation of the 5xe2x80x2-distal gene of dicistronic mRNA will be promoted by IRESmp.